The invention relates to the use of antifreeze proteins in ice confections. In particular the invention relates to the use of antifreeze proteins in ice confections to restrict the flow of flavours and/or colour.
It is a well-known problem that when an ice confection such as a water ice is consumed the flavour and colour is quickly sucked out of the product leaving essentially a block of ice which is of limited enjoyment to eat.
Further in ice confections such as water ice which are comprised of a number of different components, each having a different colour or flavour, the boundary between each component is not sharp and distinct because of the flow of the colour or flavour to a certain extent from one component to another. Consequently it has not been possible to date to provide an ice confection having thin components of a different flavour or colour which remain distinct.
WO 98/04146 (Unilever) discloses that AFPs can be incorporated into frozen food products such as ice confections to provide desirable product properties providing that the product and processing conditions are varied such that the ice crystals provided in the product have an aspect ratio of more than 1.9, preferably from 1.9 to 3.0. The specific examples in WO 98/04146 are all ice cream compositions. WO 98/04146 does not teach that it is possible to restrict the flow of colour and/or flavour in water ice products by the inclusion of an antifreeze protein in the water ice composition.
WO 96/39878 discloses a method for making a frozen composition for storage, the method not requiring a hardening step prior to storage. The frozen composition contains an antifreeze protein, in particular Type I AFP. Examples show the preparation of an aerated ice cream and an aerated frozen yoghurt. WO 96/39878 does not teach that it is possible to restrict the flow of colour and/or flavour in water ice products by the inclusion of an antifreeze protein in the water ice composition.
U.S. Pat. No. 5,118,792 (Warren et al) discloses the addition of fusion proteins, and in particular the fusion protein protein A-Saf5 into foods which are to be consumed frozen, for example, ice cream, frozen yoghurt, ice milk, sherbet, popsicles and frozen whipped cream. No examples are given where a final ice confection product is provided containing such fusion proteins. It is shown in Example 3B that when a popsicle formulation is used within the xe2x80x9csplat assayxe2x80x9d, growth of the ice crystals is restricted. U.S. Pat. No. 5,118,792 does not teach that it is possible to restrict the flow of colour and/or flavour in water ice products by the inclusion of an antifreeze protein in the water ice composition.
We have now found that the addition of antifreeze proteins to ice confections restricts the flow of flavour and/or colour.
Accordingly the invention provides the use of an antifreeze protein within an ice confection to restrict the flow of flavour or colour ions or molecules present as either solutes or a dispersion wherein the ice confection contains no protein other than the antifreeze protein.
By antifreeze protein (AFP) is meant a protein which has significant ice recrystallisation inhibition properties as measured in accordance with Example 1. The AFP provides an ice particle size upon recrystallisation of less than 20 xcexcm, more preferred from 5 to 15 xcexcm.
Preferably the ice confection comprises at least 0.0005% by weight antifreeze protein, more preferably 0.0025% by weight antifreeze protein. Typically the ice confection will comprise from 0.0005% by weight to 0.005% by weight antifreeze protein.
For some applications it may be advantageous to include a mixture of two or more different AFPs into the ice confection.
The AFP for use in products of the invention can be any AFP suitable for use in food products. Examples of suitable sources of AFP are for example given in the article xe2x80x9cAntifreeze proteins and their potential use in frozen food productsxe2x80x9d, Marylin Griffith and K. Vanya Ewart, Biotechnology Advances, vol 13, pp375-402, 1995 and in patent applications WO 98/04699, WO 98/04146, WO 98/04147, WO 98/04148 and WO 98/22591.
The AFPs can be obtained from their sources by any suitable process, for example the isolation processes as described in the above mentioned documents.
One possible source of AFP materials is fish. Examples of fish AFP materials are antifreeze glycoproteins (AFGP) (for example obtainable from Atlantic cod, Greenland cod and Tomcod), type I AFP (for example obtainable from Winter flounder, Yellowtail flounder, Shorthorn sculpin and Grubby sculpln), Type II AFP (for example obtainable from Sea raven, Smelt and Atlantic herring) and type III AFP (for example obtainable from Ocean Pout, Atlantic wolffish, Radiated shanny, Rock gunnel and Laval""s eelpout). A preferred example of the latter type is described in WO 97/02343.
Another possible source of AFP material is invertebrates. Also AFPs may be obtained from Bacteria.
A third possible source of AFP material is plants. Examples of plants containing AFPs are garlic-mustard, blue wood aster, spring oat, winter cress, winter canola, Brussels sprout, carrot, Dutchman""s breeches, spurge, daylily, winter barley, Virginia waterleaf, narrow-leaved plantain, plantain, speargrass, Kentucky bluegrass, Eastern cottonwood, white oak, winter rye, bittersweet nightshade, potato, chickweed, dandelion, spring and winter wheat, triticale, periwinkle, violet and grass.
Both naturally occurring species may be used or species which have been obtained through genetic modification. For example micro-organisms or plants may be genetically modified to express AFPs and the AFPs may then be used in accordance to the present invention.
Genetic manipulation techniques may be used to produce AFPs having at least 80%, more preferred more than 95%, most preferred 100% homology to the AFPs directly obtained from the natural sources. For the purpose of the invention these AFPs possessing this high level of homology are also embraced within the term xe2x80x9cAFDsxe2x80x9d.
The genetic manipulation techniques may be used as follows: An appropriate host cell or organism would be transformed by a gene construct that contains the desired polypeptide. The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector encoding the necessary elements for transcription and translation and in such a manner that they will be expressed under appropriate conditions (for example in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.
A number of expression systems may be utilised to express the polypeptide coding sequence. These include, but are not limited to, bacteria, yeast insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors.
A wide variety of plants and plant cell systems can be transformed with the nucleic acid constructs of the desired polypeptides. Preferred embodiments would include, but are not limited to, maize, tomato, tobacco, carrots, strawberries, rape seed and sugar beet.
For some natural sources the AFPs may consist of a mixture of two or more different AFPs.
Preferably the antifreeze protein is chosen such that it gives an aspect ratio of more than 1.9 to the ice crystal, preferably from 1.9 to 3.0, more preferably from 2.0 to 2.9, even more preferred from 2.1 and 2.8 (see WO 98/04146). Aspect ratio is defined as the maximum diameter of a particle divided by its minimum diameter. The aspect ratio can be determined by any suitable method. A preferred method is illustrated in the Examples (Example 3).
For the purpose of the invention the preferred AFPs are derived from fish. Especially preferred is the use of fish proteins of the type III, most preferred HPLC 12 as described in our case WO 97/02343.
Suitable ice confections which contain no protein other than the AFP include water ices, sorbet, granitas and frozen fruit purxc3xa9es.
Preferably the ice confection is a water ice.
By water ice is meant a frozen solution made essentially from water, sugar, fruit acid or other acidifying agent, colour, fruit or fruit flavouring.
The water ice will typically have an ice content of at least 30% by volume when measured at xe2x88x9218xc2x0 C., more preferably at least 40% by volume when measured at xe2x88x9218xc2x0 C., most preferably at least 50% by volume when measured at xe2x88x9218xc2x0 C.
The ice content may be determined following the techniques described in the article by B de Cindio and S Correra in the Journal of Food Engineering, Volume 24, pages 405-415, 1995. The enthalpy data required for this technique is obtained using adiabatic calorimetry (Holometrix Adiabatic Calorimeter). The ice contents as expressed herein are measured on an 80 g sample poured into the sample holder of the calorimeter and cooled to xe2x88x9275xc2x0 C. by placing the assembly in dry ice prior to placing in the calorimeter (pre-cooled to between xe2x88x9270xc2x0 C. and xe2x88x9280xc2x0 C.). The enthalpy data obtained was analysed to give ice content as a function of the temperature following the method of Cindio and Carrera.
In general the water ice has a total soluble solids content of less than 40% by weight, preferably less than 25% by weight, most preferably less than 15% by weight. For low calorie water ices the soluble solids content may be as low as approximately 5% by weight.
The total soluble solids content is measured at 4xc2x0 C. and is the % by weight of the total composition that is dissolved at that temperature.
The ice confection may be aerated or unaerated, preferably the ice confection is unaerated.
By unaerated is meant an ice confection having an overrun of less than 25% (equivalent to 0.2 volume fraction of air), preferably less than 10% (equivalent to 0.09 volume fraction of air). During the processing of the ice confection no deliberate steps such as whipping are undertaken to increase the gas content of the product. However, it should be realised that during normal methods for the preparation of non-aerated ice confections, low levels of gas or air may be incorporated into the product, for example due to the mixing conditions use.
Typical colour materials used in ice confections could for example include carmoisine, carotene, anthocyanins, chlorophyll, chlorophyllins, copper complexes of chlorophylls and chlorophyllins, riboflavin, riboflavin-5xe2x80x2-phosphate, caramels, vegetable carbon black, paprika extract, capsanthin, capsorubin, beetroot Red, calcium carbonate, titanium dioxide, iron oxides and hydroxides, annatto extract, curcumin, tartrazine, quinoline yellow, sunset yellow FCF, cochineal, ponceau 4R, allura red AC, patent blue V, indigo carmine, brilliant blue FCF, green S, brilliant black BN, brown HT, lycopene, beta-apo-8xe2x80x2-carotenal (C30), ethyl ester of Beta-apo-8xe2x80x2-carotenlc acid (C30) and lutein
Typical flavour materials used in ice confections could for example include natural, nature identical or synthetic flavour compounds, examples of which include; cherry, strawberry, raspberry, orange, banana, lemon, lime, lychee, guava, passion fruit, mango, grape, kiwi, melon, pineapple, papaya, apple, plum, apricot, peach, pear, mint, toffee, caramel, licorice, coffee, cotton candy and bubblegum.
The inclusion of antifreeze proteins into ice confections results in the formation of a strong, close-packed continuous network of ice crystals within the ice confection.
By close-packed continuous network of ice crystals is meant that any given ice crystal is connected to at least one other ice crystal.
In unaerated ice confections which have been frozen with agitation, the degree of network formation can be measured as contiguity.
Contiguity is defined as the ratio of the particle to particle interface area divided by the total interface area. It is thus a measure of the degree of network formation of the particle phase. Example 2 shows a method for the measurement of contiguity.
Unaerated ice confections according to the nvention have a contiguity of at least 0.2, as measured by the test given in Example 2, for an ice content of from 50-90%, preferably 54-85% by weight when measured at xe2x88x9218xc2x0 C.
In unaerated ice confections which have been frozen by any means, the degree of network formation can be measured as the Euler-Poincare characteristic of the ice phase. The Euler-Poincare characteristic is a measure of the degree of network formation of a particular phase. The lower and more negative the value of the Euler-Poincare characteristic, the greater the continuity of the phase in question. Example 4 shows a method for the measurement of the Euler-Poincare characteristic.
Unaerated ice confections according to the invention have an ice phase Euler-Poincare characteristic of less than xe2x88x92150 mmxe2x88x922, as measured by the test given in Example 4, for an ice content of from 50-90%, preferably 54-85% by weight when measured at xe2x88x9218xc2x0 C.
The use of an antifreeze protein within an ice confection to restrict the flow of flavour or colour ions or molecules present as either solutes or a dispersion provides a number of advantages.
In particular, ice confection products are provided for which the flavour and/or colour is not significantly sucked out during consumption. Flavour and/or colour is retained throughout the ice confection during the total consumption time.
A further advantage of the use of AFP to restrict the flow of flavour and/or colour is that multi-component products may be provided, each component having a different flavour and/or colour and the distinction between each comoonent remains sharp. In particular this allows produces having thin components of different colour and/or flavour to be provided. Each thin component remaining distinct from one another, there being substantially no merging of the different flavour and/or colour over time.
The ice confection according to the invention may comprise the entire product or may be included within a composite product.
For example a product may be provided having a conventional ice cream core coated with 2 or more thin layers of water ice containing AFP, each layer of water ice being a different flavour and/or colour.